Propagation of undifferentiated embryonic stem cells in hyaluronic acid hydrogel

ABSTRACT

Embryonic stem cells are propagated in a hyaluronic acid.

This application claims priority to U.S. Provisional Application No.60/692,915, filed Jun. 22, 2005, the entire contents of which areincorporated herein by reference.

This invention was made with support from the National Institutes ofHealth, K22 DE-015761, P41 EB002520-1A1 and 1R01HL076485-01A2. The U.S.government may have certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to in vitro methods for promoting the propagationof embryonic stem cells.

BACKGROUND OF THE INVENTION

Monolayer culture on a mouse or human feeder layer, Matrigel (an animalbasement membrane preparation extracted from Engelbreth-Holm-Swarm mousesarcoma), laminin, fibronectin, and in human serum are common methodsavailable today for the propagation of undifferentiated hESCs^(1,3,4,6).While these substrates have enabled much progress in HESC research,concerns remain about their undefined composition, variability betweenbatches, and the hazard of zoonosis transmitted from materials of animalorigin. Additionally, a cell monolayer is distinctly different from the3D architecture of a developing blastocyst, where hESCs are embedded inan extracellular matrix (ECM), which in turn regulates their growth anddifferentiation^(7,8). Thus, it is a desirable to promote HESCpropagation in a 3D environment.

PCT Publication WO/2006/033103 discloses the use of hyaluronicacid-laminin gels to maintain populations of embryonic stem cells invitro. However, cells encapsulated in these matrices divide into cellsexhibiting different morphologies, e.g., endothelial-like cells andepithelial-like cells. Thus, it is desirable to provide a matrix inwhich proliferating cells maintain the same morphology and phenotype.

SUMMARY OF THE INVENTION

In one aspect, the invention is a composition including a biocompatiblematrix including cross-linked hyaluronic acid and mammalian embryonicstem cells disposed within the biocompatible matrix. The composition issubstantially free of laminin. The composition may further include abiocompatible aqueous solvent. The mammalian embryonic stem cells may behuman embryonic stem cells. The hyaluronic acid may be cross-linkedthrough methacrylate moieties or through acrylate, thiol, or aminegroups, or through biotin-streptavidin interactions. A density of cellsin a composition may be from about 5 million cells/ml to about 10million cells/ml. At least 80% of the embryonic stem cells may expressone or more of tumor-rejecting antigen, stage specific embryonicantigen-4, and Oct 4. At most, 10% of the embryonic stem cells mayexpress one or more of CD31, alpha-fetoprotein, and tubulin. The cellsencapsulated within the biocompatible matrix may maintain a stablephenotype in culture for at least 30 doublings, 30 days, or 40 days. Thebiocompatible aqueous solvent may be culture media.

In another aspect, the invention is a biocompatible matrix consistingessentially of cross-linked hyaluronic acid, mammalian embryonic stemcells disposed within the biocompatible matrix, and a biocompatibleaqueous solvent, for example, culture media.

In another aspect, the invention is a composition including abiocompatible matrix comprising cross-linked hyaluronic acid, mammalianembryonic stem cells disposed within the biocompatible matrix, and abiocompatible aqueous solvent, for example, culture media. Theconcentration of the hyaluronic acid in the solvent is greater thanabout 1.5% by weight, for example, greater than about 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, or 10% by weight.

In another aspect, the invention is a method of culturing embryonic stemcells. The method includes providing a population of embryonic cells,combining the embryonic stem cells with hyaluronic acid to form amixture, and causing the hyaluronic acid to cross-link in a solvent,thereby encapsulating the embryonic stem cells in a hyaluronic acidhydrogel. The encapsulated embryonic stem cells may be cultured in invitro. The embryonic stem cells may be maintained in culture for atleast 30 days, at least 40 days, or at least 30 doublings whilemaintaining a stable phenotype. Causing may include promoting radicalchain polymerization, ionic chain polymerization or step polymerization.The method may further include allowing the cells to proliferate,releasing the cells from the hydrogel, dividing the cells into aplurality of populations, and repeating the method.

In another aspect, the invention is a method of producing a populationof embryonic stem cells. The method includes providing a population ofmammalian embryonic stem cells, combining the embryonic stem cells withmethacrylate-terminated hyaluronic acid, causing the hyaluronic acid tocross-link in a solvent, thereby encapsulating the embryonic stem cellsin a hyaluronic acid hydrogel, and contacting the hydrogel withhyaluronidase to release the embryonic stem cells.

BRIEF DESCRIPTION OF THE DRAWING

The invention is described with reference to the several figures of thedrawing, in which,

FIG. 1: HA plays a role during hESC culture on MEFs. A. Mouse embryonicfibroblasts (MEFs) secreted HA into culture medium, at concentrationsthat were over eight times higher than those measured for normal hESCgrowth medium. B. Staining of H1 hESCs grown on MEFs for HA binding site(green), undifferentiated membrane marker-TRA-1-81 (red) and nuclei(blue), revealed: (i & ii) intracellular localization of HA, including(iii) perinuclear areas (arrows) and nuclei (asterisks), as well as (iv)nucleoli (arrowheads). C. The majority of undifferentiated hESCs werefound to express HA receptors CD44 (82%; middle) and CD168 (90%; right).D. i-ii. Using immunofluorescence staining, undifferentiated HESCcolonies were easily detected using undifferentiated cell markers Oct4(green) and CD44 or CD168 (red) respectively (nuclei —blue). iii-iv.Higher magnification revealed intracellular expression of CD44 andeither membrane or intracellular expression of CD168.

FIG. 2: Encapsulation in HA hydrogels supported hESC viability andpropagation. A. Undifferentiated H9 hESCs were passaged and re-culturedon feeder layers for 4 days in culture medium containing:. (i) nomacromer, (ii) 10 μ/ml macromer, or (iii) 50 μl/ml macromer. Toxiceffects were detected only at the macromer concentration of 50 μl/ml(some damaged cells marked with arrowheads). (iv) XTT assay revealed noeffect of macromer on cell viability at a concentration of 10 μl/ml anda slight decrease in HESC viability at a macromer concentration of 50μl/ml. Results are presented with ±SD (*P<0.05). B-C. Colony arrangementof undifferentiated cells detected using light microscopy at low andhigh magnification, respectively. D-E. Incubation with XTT revealedorange dye in viable H13 hESCs. F-H. Histology sections of H9 HESC-HAconstructs cultured for 20 days demonstrate typical morphology (H&Estain) of undifferentiated colonies within 3D networks. I. i-iiFluorescence staining of H9 HESC-HA constructs cultured for 25 daysdemonstrates the presence of undifferentiated hESCs. J. Staining forKi-67 reveled that the majority of cells were proliferating. K.Caspase-3 expression was rare and L. could be observed mainly in wholecolonies undergoing apoptosis. Bars-A-E,I, K−L=100 μm; F−H, J=25 μm.

FIG. 3. hESC growth rate in HA vs. Matrigel. H9 hESCs were removed fromthe feeder layer and cultured in the same cell concentration (per area)either within a HA hydrogel or on Matrigel, in MEF conditioned medium.XTT assay show comparable rate growth during the first 4 days of culturein both systems. Results are presented with ±SD.

FIG. 4: Cell release from hydrogels and cell karyotyping. H13 hESCsgrown on MEFs were incubated for 24 hr in A. growth medium; B. 1%collagenase in growth medium; C. 1000 U/ml, and D. 2000 U/mlhyaluronidase in growth medium. To release hESCs from HA hydrogel,constructs were incubated 2000 U/ml hyaluronidase in growth medium: E.After 18 hr, small particles of hydrogels remained that trapped hESCs.F. After 24 hr, hESCs colonies were completely released from thehydrogel. G. H9 hESCs released from the hydrogel were cultured on MEFsand formed small undifferentiated colonies after 24 hours. H. H9 hESCsreleased from hydrogels were propagated on MEFs for 3 passages. Geneticintegrity was further examined and abnormalities events could not bedetected in: I. H9 p22 grown on MEFs, J. H9 p22 grown on MEFs andexposed to UV for 10 min, K. H9 p22 grown on MEFs and incubated withgrowth medium containing 2000 U/ml hyaluronidase. L. H9 p38 removed fromMEFs and encapsulated in HA hydrogels for 5 days followed by incubationwith growth medium containing 2000 U/ml hyaluronidase for 24 handre-culture on MEFs for 3 passages. Bars=100 μm

FIG. 5. EB differentiation. H13 hESCs encapsulated in HA hydrogel for 30days were released and cultured in suspension to allow EB formation.Histology sections of 30 days old EBs revealed A. typical EBorganization with B. detectable remains of HA entrapped within thebodies (arrows). C. Various cell types and cellular organization couldbe observed using higher magnification.

FIG. 6. Genetic integrity. Karyotyping of hESC H13 line was alsoevaluated in: A. H13 p25 grown on MEFs, B. H13 p25 grown on MEFs andincubated with growth medium containing 2000 U/ml hyaluronidase, and C.H13 p25 grown on MEFs and exposed to UV for 10 min. Abnormality eventscould not be detected in any of the conditions.

FIG. 7: HA Internalization and degradation. A. Encapsulation of H13hESCs in HA hydrogels was compared to dextran hydrogels after 15 days ofculture. Light microscope images of both cultures at low and highmagnifications and H&E staining of sectioned gels demonstrate embryoidbody formation in dextran gels vs. colony arrangements ofundifferentiated hESCs in HA gels. B. Undifferentiated H1 hESCs grown onMEFs were incubated overnight with fluorescein-HA, and further stainedfor CD44 and CD168. HA uptake by H9 hESC colonies: i edge of colonyinternalizing FL-HA via CD44; ii & iii intracellular localization ofFL-HA in hESC colonies C. H13 hESC colonies grown on MEFs positive forOct4 (green) express (i) Hyal 1 or (ii) Hyal 2 (red; nuclei in blue)mainly in densely packed areas of the colonies D. RT-PCR analysisrevealed high expression levels of a hyaluronidase isomer, Hyal 2, inundifferentiated H9 hESCs. PC3 line served as positive control. Bars=100μm

FIG. 8. HA receptors in response to addition of human FL-HA. FL-HA wasadded to the growth medium of H9 hESCs cultured on MEFs. Confocalanalysis revealed localization in cell membranes of both A. CD44 and B.CD168 (red, nuclei in blue).

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

In an exemplary embodiment, mammalian embryonic stem cells (ESC) aredisposed within a cross-linked hyaluronic acid matrix and cultured inappropriate media. The cells remain undifferentiated in vitro forextended periods of time. The ESC may be human or non-human ESC.

In one embodiment, ESC were encapsulated in a hydrogel scaffold that iscomposed of biologically recognized molecules. Hydrogels were selectedbecause they not only have a high water content to promote cellviability, but they are structurally and mechanically similar to thenative ECM of many tissues⁹. HA, a nonsulfated linear polysaccharide of(1-β-4) D-glucuronic acid and (1-β-3) N-acetyl-D-glucosamine, wasselected because it co-regulates gene expression, signaling,proliferation, motility, adhesion, metastasis, and morphogenesis¹⁰.Notably, HA content is greatest in undifferentiated cells and duringearly embryogenesis and then decreases at the onset ofdifferentiation¹¹. In spite of its known role in embryogenesis^(10,11),HA has not been extensively utilized for the cultivation of hESCs.

We observed that mouse embryonic fibroblasts (MEFs) that form feederlayers for HESC cultivation produce high levels of HA (FIG. 1A), andthat abundant HA binding sites were located intracellularly inundifferentiated hESCs (FIG. 1B). This is consistent with previousevidence that HA is localized intracellularly, in endosomes andperinuclear tubular vesicles¹², rough endoplasmic reticulum¹⁴, nucleiand nucleoli¹⁴. Also, without being bound by any particular theory, thesuccess of mouse feeder layers for the cultivation of hESCs might berelated to their ability to secrete HA.

During development, cellular interactions with HA are mediated at leastin part by CD44 and CD168. CD44 is a mediator for HA-induced cellproliferation and survival pathways¹⁰ and is present in human cumuluscells, oocytes, early embryos and pre-hatched blastocysts¹⁵. CD44 isalso involved in the initial binding of HA to the cell surface prior toits internalization and degradation by acid hydrolases. CD168 isinvolved in HA-induced cell locomotion¹⁶, and its expression in earlyembryos was recently documented¹⁷. During in vitro culture,undifferentiated hESCs expressed high levels of CD44 and CD168 (FIG.1C). In fact, hESC colonies cultured on MEFs could be easily visualizedby staining for CD44 (FIG. 1Di) or CD168 (FIG. 1Dii). Undifferentiatedcells were characterized by intracellular expression of CD44 (FIG.1Diii) and either membrane or intracellular expression of CD168 (FIG.1Div).

Various attempts to culture HESC monolayers on a HA substrate resultedin low efficiency of cell adhesion (<15%), consistent with low adhesionof other cells expressing HA receptors such as NIH3T3¹⁹. We thereforeelected to encapsulate ESCs within HA, in a manner that combines cellscaffolding with the use of a chemically defined system. We furtherselected HA hydrogels because they allow gentle entrapment ofdifferentiated mammalian cells without a loss of their viability²⁰. Inour previous studies, a HA hydrogel fabricated from a 2 wt % solution ofa 50 kDa macromer supported the highest viability of differentiatedmammalian cells²⁰. One advantage of HA hydrogels is that the chemistryof the network is easily controlled via reaction conditions and isuniform between different batches²⁰, in contrast with naturally derivedmatrices such as Matrigel.

To form hydrogels, HA may be chemically modified with methacrylategroups that, in the presence of light and a photoinitiator, undergo afree-radical polymerization. Exemplary initiators include but are notlimited to 2-methyl-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone(Irgacure 2959, 12959), a photoinitiator, and thermal and redoxinitiation systems such as those employed for methacrylate bone cements.The initiator may be optimized to minimize its chemical influence on theencapsulated cells and to minimize any effect that the initiationconditions may have on the cells. Other functional groups that may beused to crosslink the HA are familiar to those of skill in the art andinclude but are not limited to acrylates, amines, and thiols. Whilelight-initiated radical polymerization may provide simpler initiationand reaction conditions, other polymerization mechanisms, e.g., thermalor other radical initiation conditions, ionic chain polymerization orstep polymerization, may be employed as well. One skilled in the artwill be familiar with appropriate reactive groups, initiators, etc. forforming cross-linking polymers using these methods. Further examples maybe had by reference to Odian, Principles of Polymerization, FourthEdition, Wiley Interscience, 1994, the entire contents of which areincorporated by reference. HA may also be functionalized with biotin orstreptavidin and crosslinked through streptavidin-biotin interactions.

For encapsulation, ESCs are suspended in a solution of HA macromer andpolymerized into a network. Exemplary molecular weights of the HAmacromer range from about 5 kDa to about 2000 kDa, for example, about 50kDa, about 350 kDa, or about 1100 kDa. Exemplary molecular weights mayrange from about 5 kDa to about 50 kDa, from about 50 kDa to about 100kDa, about 100 kDa to about 500 kDa, about 500 kDa to about 1000 kDa,about 1000 kDa to about 1500 kDa, or about 1500 kDa to about 2000 kDa.The crosslink density of the resulting gels may be correlated to theinitial concentration of the HA (wt %) in the precursor solution.Exemplary concentrations may range as low as 0.5%, for example, about0.5% to about 1%, about 1% to about 2%, about 2% to about 4%, about 4%to about 6%, about 6% to about 8%, about 8% to about 10%, or evengreater. While concentrations as high as 40% may be achievable, one ofskill in the art will recognize that the concentration may be optimizedto maximize cell viability while maintaining the structural integrity ofthe hydrogel as the cells propagate.

Exemplary hydrogels fabricated with 50 kDa HA contained spatiallyuniform cell distributions (FIG. 2B-C). The viability of hESCs wasmaintained throughout the 25 days of cultivation, as demonstrated by XTTstaining (FIG. 2D-E). A typical undifferentiated morphology was observedin hESC colonies within the HA networks (FIG. 2F-H). High cellconcentrations, in the range of 5 to 10 million cells per ml of theprecursor solution, were optimal for high viability and sustained growthof hESCs. At hESC concentrations greater than ten million cells/ml,large clumps of cells formed that underwent rapid apoptosis, whereascell concentrations lower than 5 million cells per ml could not supportcolony formation within the networks (data not shown). The samephenomenon of colony concentration-dependence of hESC culture is wellrecognized for 2D monolayers^(22,23). Early in culture, the growth ratesof hESCs within HA hydrogel and on Matrigel were comparable (FIG. 3).

Exemplary HESC populations (see examples) were propagated in gels formedfrom 50 kDa HA for up to 30 doublings (˜40 days); further expansion maydepend on the hydrogel structure. For more loosely crosslinked hydrogels(e.g., 1 wt % solutions of macromer), the gel does not maintain itsstructural integrity past this point. With more densely crosslinkedhydrogels (e.g., 2 wt % solutions), the cell expansion rates arehindered. One skilled in the art will recognize that the concentrationof macromer in solution may be adjusted to optimize the propagation rateand the number of doublings. After a certain period of time, cells mayhave proliferated sufficiently that there is no more room for furthercell proliferation. This time period will vary with crosslink densitybut is around 20 days for 2 wt % solutions of 50 kDa HA. At this point,proliferation may be continued by releasing the cells from the HA geland re-encapsulating them. The optimal frequency of release andre-encapsulation (e.g., “3D passaging”) depends in part on the originalseeding density and the molecular weight and cross-link density of theHA. In some embodiments, cells may be passaged every 10 days, every 15days, every 20 days, or at some other frequency.

In one embodiment, the developing HESC colonies expressed high levels ofstem cell markers after more than 30 days of culture, including thetumor rejecting antigen (TRA)-1-81 (˜93%), stage specific embryonicantigen-4 (SSEA-4) (˜98%), and Oct 4 (˜97%); (FIG. 21). For example, atleast 80%, at least 85%, at least 90%, or at least 95% of ESC in culturemay express one of these markers, indicating that the cells aremaintaining the stem cell phenotype. For cells cultured according to anexemplary embodiment of the invention, differentiation markers formesoderm (CD31), endoderm (α-fetoprotein) and ectoderm (tubulin) werenot detected. In some embodiments, at most 10%, at most 5%, or at most1% of the ESC express one or more of these markers.

The maintenance of cellular viability within the hydrogel constructs wasdocumented by the presence of an array of markers. The human Ki-67protein, which is present during all active phases of the cell cycle,was expressed by the majority of encapsulated hESCs (58±5%) (FIG. 2J).Only occasional apoptotic events could be observed using a Tunnel assay(14±3%). These results correlate to a recent study which showedthat >50% of the cell nuclei within hESC colonies grown on MEFs are in aproliferating phase²⁴. Only rare expression of caspase-3, a markeractivated in cells undergoing apoptosis, was found within HA-HESCconstructs (3±8%). When detected, caspase-3 appeared in a whole colonyrather than in single cells within different colonies (FIG. 2K-L).Therefore, under the conditions studied, diffusion of nutrients andoxygen to the cells through the 2 wt % HA hydrogel appeared to be rapidenough to support normal cell growth rates.

To use the ESC for research and cellular therapy, the cells may need tobe released from the hydrogel. An exemplary method for releasing thecells is by treating the HA hydrogel with hyaluronidase²⁰ at aconcentration of about 500 to about 2000 U/mL. We examined hESCs todetermine if they survive long-term treatment (i.e., 24 hours) withhyaluronidase. HESC colonies incubated with growth medium containinghyaluronidase at all concentrations preserved their normal morphologywith no apparent loss of viability (FIG. 4A-D). We found thathyaluronidase concentrations of <1000 U/ml resulted in only partialdegradation of HA hydrogels over a 24 hr period, and were associatedwith low efficiencies of HESC retrieval. Incubation of HA-HESCconstructs in growth medium containing 2000 U/ml hyaluronidase resultedin complete degradation of the hydrogel (FIG. 4E-F). One of skill in theart will recognize that the optimal concentration and incubation timemay vary depending on the crosslink density and molecular weight. Lowerconcentrations may also be employed where it is desirable to study thegel after the cells are released. Colonies released from the gelsreadily adhered to the MEF (FIG. 4G) with high adherence efficiency (80%after 48 hours) and proliferated for at least 5 passages without thedifferentiation that is often seen in standard monolayer cultures ofhESCs (FIG. 4H).

Importantly, the release of hESCs from the HA hydrogels was associatedwith the preservation of cell viability and undifferentiated state. Incorrelation with the in situ staining, FACS analysis showed high levelsof expression of stem cell marker SSEA-4 (96%) and minimal levels of thedifferentiation marker CD31 (<0.3%) (data not shown). To demonstratethat the HA-borne hESC colonies maintained their pluripotency, thedifferentiation potential of the cells released from HA hydrogels wasexamined by the spontaneous formation of embryoid bodies (EBs). hESCsencapsulated in HA hydrogels for 35 days, released using hyaluronidaseand cultured in suspension formed EBs containing various cell types(FIG. 5).

The proposed system for ESC culture in a three-dimensional HA hydrogeland the release of expanded ESCs involves the exposure of ESCs to lowintensity UV light (e.g., ˜10 mW/cm² for 10 min) and treatment withhyaluronidase (e.g., 2000 U/ml for 24 hours). Since these factors couldpotentially affect the genetic integrity of ESCs, karyotype analysis wasperformed on: (i) undifferentiated hESCs cultured on MEFs (H9 line p22;H13 line p25); (ii) undifferentiated hESCs cultured on MEFs (H9 linep22; H13 line p25) and exposed to UV light for 10 min; (iii)undifferentiated hESCs cultured on MEFs (H9 line p22; H13 line p25)treated with hyaluronidase (2000 U/ml) for 24 h; and (iv)undifferentiated hESCs (H9 line p38) encapsulated in HA gels for 5 daysfollowed by their release and re-culture on MEFs for an additional 3passages. All hESCs were found to have normal 46XX karyotyping (FIG.4I-L and FIG. 6). Hence, the application of UV light and hyaluronidaseat the levels necessary for releasing cells from HA gels appear to besafe for hESCs.

To determine the importance of ESC-HA interactions for the propagationof ESCs in their undifferentiated state, hESCs were encapsulated innetworks formed from a different polysaccharide, dextran, using theexact same methodology of photopolymerization for cell encapsulation. Incontrast to the proliferation of undifferentiated hESC colonies in theHA system, dextran hydrogels induced hESC differentiation and theformation of embryoid bodies (FIG. 7A). These results are consistentwith data published for other hydrogel systems that also supported hESCdifferentiation^(25,26). Without being bound by any particular theory,the regulatory role of HA in the maintenance of hESCs in theirundifferentiated state, in vitro as well as in vivo, may contribute tothe ability of hESCs to propagate in HA without differentiating.

The addition of human FL-HA to the culture of hESCs on MEFs resulted inthe localization of HA receptors to the cell membranes, first at theedges of cell colonies and then at their centers (see FIG. 8). FL-HA wasinternalized through the membrane receptors (FIG. 7Bi) and localizedwithin the cells (FIG. 7Bii-iii), indicating receptor-mediatedinternalization of HA by hESCs. Immunofluorescence of HESC coloniescultured on MEF revealed that densely packed colonies expressed humanhyaluronidase Hyal 1&2 (FIG. 7C). RT-PCR analysis corroborated thathESCs express high levels of expression of Hyal 2, one of the isoformsof human hyaluronidase (FIG. 7D). It was previously suggested that HAoriginates from the pericellular material which is degradedintracellularly^(27,28). Without being bound by any particular theory,our data suggest that hESCs are able to uptake and degrade HA andthereby remodel HA gels, which can further promote their survival andproliferation.

These data demonstrate that viable, proliferating ESCs can be maintainedin their undifferentiated state when cultured in HA hydrogels, andreleased without the loss of cell viability. ESC survival andproliferation were associated with the presence of developmentallyrelevant signals and the ability of cells to remodel the HA gel.Hydrogels formed as networks of HA fibers appear to have the ability tomaintain proliferating hESCs in their undifferentiated state (incontrast to dextran networks or cell monolayers on HA) and underchemically defined conditions (in contrast to soluble HA, MEFs, Matrigeland human serum).

EXAMPLES

hESCs. Three different lines of hESCs were studied (WA9, WA13 and WA1,obtained from WiCell Research Institute, Madison, Wis.; p19-40).

hESC culture on MEFs. hESCs were grown on inactivated mouse embryonicfibroblasts (MEFs) in growth medium including 80% KnockOut DMEM,supplemented with 20% KnockOut Serum Replacement, 4 ng/ml basicFibroblast Growth Factor, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1%non-essential amino acid stock (Invitrogen Corporation, Carlsbad,Calif.). hESCs were passaged every four to six days using 1 mg/ml typeIV collagenase (Invitrogen Corporation, Carlsbad, Calif.).

hESC encapsulation and release. Methacrylated HA was synthesized aspreviously described²⁰. Briefly, HA (50 kDa, Lifecore) was dissolved indeionized water and adjusted to a pH of 8.0 with 5 N NaOH. Methacrylicanhydride (Aldrich) was slowly added and the reaction mixture wasincubated overnight at room temperature. The product was dialyzed forpurification, lyophilized, and stored as a powder at 0° C. Themethacrylated HA was dissolved at a concentration of 2 wt % in PBScontaining 0.05 wt %2-methyl-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure2959, I2959) and hESCs were added (0.5−1×10⁷ cells/ml precursorsolution). The mixture was pipetted into a sterile mold (50 μL volumeper well, to obtain discs measuring 3 mm in diameter×2 mm thick), andphotopolymerized (˜10 mW/cm² UV light, BlakRay, for 10 min).

Dextran-acrylate macromer was prepared as described previously²⁹.Dextran (10 g) and vinyl acrylate (1.21 g) were dissolved in DMSO (150mL) and the reaction initiated by adding 1.5 g of Proleather (enzymefrom Bacillus sp.). The reaction mixture was shaken at 50° C. (250 rpm)for 72 h, and then precipitated in acetone. The precipitate wasdissolved in water and dialyzed for 5 days against milli-Q water, at 420C., and finally lyophilized. hESCs were encapsulated within the dextranusing the same procedures as for HA hydrogels.

Cell-gel constructs were cultivated in hESC grwoth medium containing 100ng/ml bFGF or MEF conditioned media as previously described¹ Briefly,hESCs growth medium was incubated on inactivated MEF for 24 hours andcollected and filtrated. bFGF (4 ng/ml) was added before use. Constructswere incubated up to 40 days. To release encapsulated hESCs, HAconstructs were incubated for 24 h in hESC growth medium containing 100,500, 1000 or 2000 U/ml hyaluronidase (Sigma, St. Louis, Mo.). Forre-culture, cells were collected, centrifuged, washed three times withPBS to remove any hydrogel residues, re-suspended in growth medium, andcultured on MEF coated dishes using standard methods^(22,23). For EBformation, hESCs were cultivated in non-adherent Petri-dishes.

Presence of HA in medium. MEF conditioned medium was prepared aspreviously described¹ and compared to hESCs growth medium with respectto the levels of HA using a HA test kit (Corgenic, Inc., Westminster,Colo.) according to the manufacturer's instructions.

HA receptors and stem cell/differentiation markers. hESCs were removedfrom MEFs or released from hydrogels and filtered through a 40 μm meshstrainer (BD, San Jose, Calif.). Expression of alkaline phosphatase (AP)was considered as an indicator of undifferentiated state of hESCs.Intrastain kit (Dako California Inc. Carpinteria, Calif.) was used forthe fixation and permeabilization of cell suspensions, according to themanufacturer's instructions. Briefly, dissociated hESCs were blockedwith 5% FBS/PB-S, incubated with anti-human CD44 clone A3D8 (Sigma, StLouis, Mo.), or IgG antibody (R&D systems, Minneapolis, Minn.) for 30min, and washed with PBS, followed by incubation with donkey anti-mouseFITC (Vector Labs Burlingame, Calif.) for 15 min. Cells were stainedwith APC conjugated anti-human AP or PE conjugated anti-human SSEZ4(both from R&D systems, Minneapolis, Minn.) for stem cell markers orwith FITC conjugated anti-human CD31 (BD, San Jose, Calif.) as marker ofdifferentiation. The cells were washed twice prior to flow cytometry.hESCs were analyzed using FACSCalibur (BDIS) and Cell Quest software(BDIS).

Toxicity Assay

Because hESCs can be sensitive to culture conditions²¹, we assessed anytoxicity of the methacrylated HA macromer. hESCs were propagated inmonolayers with two concentrations (i.e., 10 and 50 μ/ml) of the HAmacromer in the culture media. Colonies of hESCs were formed at allculture conditions and have continuously grown with time (FIG. 2Ai-iii). Comparison of the proliferation rates revealed toxic effectsonly at a macromer concentration of 50 μ/ml (FIG. 2A iv), a levelcorresponding to completely non-polymerized HA and, therefore muchhigher than that seen by the encapsulated cells. The rate of cellproliferation at a macromer concentration of 10 μ/ml, a levelcorresponding to a HA hydrogel that was polymerized to 80% incorporationof the macromer, was indistinguishable from cells cultured in controlmedium (FIG. 2A iv). Radical polymerization to produce looselycrosslinked HA hydrogels occurs at high conversion rates and the releaseof unreacted macromer is only minimal, thus eliminating any toxicitythat may result from the presence of free HA macromer.

Proliferation assay. Proliferating cells were detected by the XTT kit(Sigma, St. Louis Mo.) according to the manufacturer's instructions.Undifferentiated hESCs cultured in the presence of macromer on Matrigeland within HA cultures were incubated for 4 h in medium containing 20%(v/v) XTT solution. For analysis, 150 μl of the medium were removed,placed in a 96-plate well and read in a microplate reader at 450 nm. XTTwas also used for visual analysis of viable cells within hydrogels inwhich HA constructs were incubated for 4 h in medium containing 20%(v/v) XTT solution and examined using Inverted light microscopy (NikonDiaphot system).

Immunohistochemistry. HA constructs were either embedded in histo-gel ordirectly fixed in 10% neutral-buffered formalin (Sigma, St. Louis, Mo.)overnight, dehydrated in graded alcohols (70-100%), embedded inparaffin, sectioned to 4 μm, and stained with hematoxylin/eosin.Immunostaining was performed using a Dako LSAB®+ staining kit (DakoCalifornia Inc. Carpinteria, Calif.) with specific anti tumor rejectionantibody (TRA)-1-60, anti TRA-1-81, and anti CD44 clone P3H9 (ChemiconTemecula, Calif.). Mouse IgG isotype-matching (R&D systems, Minneapolis,Minn.) or secondary antibody alone (from Dako LSAB®+ staining kit)served as negative controls. For proliferation assessment, anti-Ki67 (BDPharmingen, San Jose, Calif.) was used. For apoptotic assessment, tunnelassay (Roche Applied Science, Indianapolis, Ind.) was preformedaccording to the manufacturer's instructions and sections were stainedfor anti-Caspase-3 (Cell Signaling, Beverly, Mass.). For quantification,3 gels were scored for positive cells.

Immunofluorescence and confocal microscopy. hESC colonies grown on MEFsand HA-hESCs constructs were fixed in situ with accustain (Sigma, StLouis, Mo.) for 20-25 min at room temperature. After blocking with 5%FBS, cells were stained with one of the following primary antibodies:anti-human SSEA4, anti-TRA-1-60, anti-TRA-1-81, anti-Oct3/4, anti-CD44clone P3H9, anti-Tubulin III isoform (all from Chemicon Temecula,Calif.), anti-CD44 clone A3D8 (Sigma, St Louis, Mo.), anti-CD168 (NovoCastra, Newcastle upon Tyne, UK), anti-CD31, anti-α-fetoprotein (DakoCalifornia Inc. Carpinteria, Calif. ), anti-Hyal 1 and Hyal 2 (kindlyprovided by Inna Gitelman from Ben-Gurion University of the Negev,Israel). Cells were then rinsed three times with PBS (Invitrogencorporation, Carlsbad, Calif.) and incubated for 30 min with suitableFITC-conjugated (R&D systems, Minneapolis, Minn.) or Cy3-conjugated(Sigma, St Louis, Mo.) secondary antibodies. DAPI (2 μg/ml; Sigma, StLouis, Mo.) or To-pro 3 (1:500; Molecular Probe, Invitrogen corporation,Carlsbad, Calif.) were added during the last rinse. IgG isotype-matchingusing mouse or goat (both from R&D systems, Minneapolis, Minn.) orsecondary antibody alone served as controls. The immuno-labeled cellswere examined using either fluorescence microscopy (Nikon TE300 invertedmicroscope) or confocal laser scanning microscopy (Zeiss LSM510 Laserscanning confocal).

HA binding and uptake. The binding assay of fluorescein-labeledhyaluronan was performed as previously described¹⁴. Briefly, hESCs werecultured on coverslips. After gentle washing, human fluorescein-labeledhyaluronan (100 μg/ml, Sigma, St Louis, Mo.) was added to the growthmedium for 16 h at 4° C. Following three washes with ice-cold PBS, thecells were fixed in 100% ice-cold acetone for 10 min, air-dried, andthen rehydrated 15 min in PBS. Processed cells were further stained withanti-CD44 or anti-CD168 and examined.

RT-PCR. Total RNA was extracted using TriZol® (Gibco Invitrogen Co., SanDiego, Calif.), according to manufacturer's instructions. Total RNA wasquantified by a UV spectrophotometer and 1 μg was used for each RTsample. One step RT-PCR kit (Qiagen Inc, Valencia, Calif.) was usedaccording to manufacturer's instructions. RT reaction mix was used fornegative controls. PCR conditions consisted of: 5 min at 94° C. (hotstart), 30-40 cycles (actual number noted below) of: 94° C. for 30 sec,annealing temperature (Ta, noted in Table 1) for 30 sec, 72° C. for 30sec. A final 7 min extension at 72° C. was performed. Primers usedinclude: HYAL 1 sense 5′GGGCACCTACCCCTACTACACG3′, antisense5′CATCTGTGACTTCCCTGTGCC3′; HYAL2 sense 5′TGGCCCACGCCTCAAGGTGCC3′,antisense 5′GGCCATGGAGGGCGGAAGCA3′; HYAL3 sense5′AGCACACTGTGAGGCCCGCTTT3′, antisense 5′GGGGATGTCGGTGCCCAACAA3′; PH205′CTTAGTCTCACAGAGGCCAC3′, 5′TACACACTCCTTGCTCCTGG3′. The amplifiedproducts were separated on 2% agarose gels containing ethidium bromide.

Karyotyping analysis. Cells were prepared and analyzed as previouslydescribed and recommended³⁰. Karyotyping analysis was performed by DanaFaber /Harvard Cancer Research Center, Cytogenetics Laboratory,Cambridge, Mass.

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Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

1. A composition comprising: a biocompatible matrix comprising cross-linked hyaluronic acid; and mammalian embryonic stem cells disposed within the biocompatible matrix, wherein the composition is substantially free of laminin.
 2. The composition of claim 1, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 0.5% to about 1% by weight.
 3. The composition of claim 1, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 1% to about 2% by weight.
 4. The composition of claim 1, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 2% to about 4% by weight.
 5. The composition of claim 1, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 4% to about 6% by weight.
 6. The composition of claim 1, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 6% to about 8% by weight.
 7. The composition of claim 1, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 8% to about 10% by weight.
 8. The composition of claim 1, wherein the mammalian embryonic stem cells are human embryonic stem cells.
 9. The composition of claim 1, wherein the hyaluronic acid is cross-linked through methacrylate moieties.
 10. The composition of claim 1, wherein the hyaluronic acid is crosslinked through acrylate, thiol, or amine groups or through biotin-streptavidin interactions.
 11. The composition of claim 1, wherein the density of cells in the composition is from about 5 million cells/mL to about 10 million cells/mL.
 12. The composition of claim 1, wherein at least 80% of the embryonic stem cells express one or more of tumor rejecting antigen (TRA), stage specific embryonic antigen-4 (SSEA-4), and Oct
 4. 13. The composition of claim 1, wherein at least 85% of the embryonic stem cells express one or more of tumor rejecting antigen (TRA), stage specific embryonic antigen-4 (SSEA-4), and Oct
 4. 14. The composition of claim 1, wherein at least 90% of the embryonic stem cells express one or more of tumor rejecting antigen (TRA), stage specific embryonic antigen-4 (SSEA-4), and Oct
 4. 15. The composition of claim 1, wherein at least 95% of the embryonic stem cells express one or more of tumor rejecting antigen (TRA), stage specific embryonic antigen-4 (SSEA-4), and Oct
 4. 16. The composition of claim 1, wherein at most 10% of the embryonic stem cells express one or more of CD31, alpha-fetoprotein, and tubulin.
 17. The composition of claim 1, wherein at most 5% of the embryonic stem cells express one or more of CD31, alpha-fetoprotein, and tubulin.
 18. The composition of claim 1, wherein at most 1% of the embryonic stem cells express one or more of CD31, alpha-fetoprotein, and tubulin.
 19. The composition of claim 1, wherein the cells encapsulated within the biocompatible matrix maintain a stable phenotype in culture at least 30 doublings.
 20. The composition of claim 1, wherein the cells encapsulated within the biocompatible matrix maintain a stable phenotype in culture for at least 30 days.
 21. The composition of claim 1, wherein the cells encapsulated within the biocompatible matrix maintain a stable phenotype in culture for at least 40 days.
 22. The composition of claim 1, wherein the molecular weight of the hyaluronic acid is about 50 kDa, about 350 kDa, or about 1100 kDa.
 23. The composition of claim 1, wherein the molecular weight of the hyaluronic acid is from about 5 kDa to about 50 kDa.
 24. The composition of claim 1, wherein the molecular weight of the hyaluronic acid is from about 50 kDa to about 100 kDa.
 25. The composition of claim 1, wherein the molecular weight of the hyaluronic acid is from about 100 kDa to about 500 kDa.
 26. The composition of claim 1, wherein the molecular weight of the hyaluronic acid is from about 500 kDa to about 1000 kDa.
 27. The composition of claim 1, wherein the molecular weight of the hyaluronic acid is from about 1000 kDa to about 1500 kDa.
 28. The composition of claim 1, wherein the molecular weight of the hyaluronic acid is from about 1500 kDa to about 2000 kDa.
 29. The composition of claim 1, wherein the biocompatible aqueous solvent is culture media.
 30. A composition, comprising: a biocompatible matrix consisting essentially of cross-linked hyaluronic acid; mammalian embryonic stem cells disposed within the biocompatible matrix; and a biocompatible aqueous solvent.
 31. The composition of claim 30, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 0.5% to about 1% by weight.
 32. The composition of claim 30, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 1% to about 2% by weight.
 33. The composition of claim 30, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 2% to about 4% by weight.
 34. The composition of claim 30, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 4% to about 6% by weight.
 35. The composition of claim 30, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 6% to about 8% by weight.
 36. The composition of claim 30, further comprising a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid is from about 8% to about 10% by weight.
 37. The composition of claim 30, wherein the mammalian embryonic stem cells are human embryonic stem cells.
 38. The composition of claim 30, wherein the hyaluronic acid is crosslinked through methacrylate moieties.
 39. The composition of claim 30, wherein the hyaluronic acid is crosslinked through acrylate, thiol, or amine groups or through biotin-streptavidin interactions.
 40. The composition of claim 30, wherein the density of cells in the composition is from about 5 million cells/mL to about 10 million cells/mL.
 41. The composition of claim 30, wherein at least 80% of the embryonic stem cells express one or more of tumor rejecting antigen (TRA), stage specific embryonic antigen-4 (SSEA-4), and Oct
 4. 42. The composition of claim 30, wherein at most 10% of the embryonic stem cells express one or more of CD31, alpha-fetoprotein, and tubulin.
 43. The composition of claim 30, wherein the cells encapsulated within the biocompatible matrix maintain a stable phenotype in culture at least 30 doublings.
 44. The composition of claim 30, wherein the cells encapsulated within the biocompatible matrix maintain a stable phenotype in culture for at least 30 days.
 45. The composition of claim 30, wherein the cells encapsulated within the biocompatible matrix maintain a stable phenotype in culture for at least 40 days.
 46. The composition of claim 30, wherein the molecular weight of the hyaluronic acid is about 50 kDa, about 350 kDa, or about 1100 kDa.
 47. The composition of claim 30, wherein the molecular weight of the hyaluronic acid is from about 5 kDa to about 2000 kDa.
 48. The composition of claim 30, wherein the biocompatible aqueous solvent is culture media.
 49. A composition comprising: a biocompatible matrix comprising cross-linked hyaluronic acid; mammalian embryonic stem cells disposed within the biocompatible matrix; and a biocompatible aqueous solvent, wherein the concentration of the hyaluronic acid in the solvent is greater than 1.5% by weight.
 50. The composition of claim 49, wherein the concentration is greater than about 2.0% by weight.
 51. The composition of claim 49, wherein the concentration of the hyaluronic acid is greater than about 3% by weight.
 52. The composition of claim 49, wherein the concentration of the hyaluronic acid is greater than about 4% by weight.
 53. The composition of claim 49, wherein the concentration of the hyaluronic acid is greater than about 5% by weight.
 54. The composition of claim 49, wherein the mammalian embryonic stem cells are human embryonic stem cells.
 55. The composition of claim 49, wherein the hyaluronic acid is crosslinked through methacrylate moieties.
 56. The composition of claim 49, wherein the hyaluronic acid is crosslinked through acrylate, thiol, or amine groups or through biotin-streptavidin interactions.
 57. The composition of claim 49, wherein the density of cells in the composition is from about 5 million cells/mL to about 10 million cells/mL.
 58. The composition of claim 49, wherein at least 80% of the embryonic stem cells express one or more of tumor rejecting antigen (TRA), stage specific embryonic antigen-4 (SSEA-4), and Oct
 4. 59. The composition of claim 49, wherein at least 85% of the embryonic stem cells express one or more of tumor rejecting antigen (TRA), stage specific embryonic antigen-4 (SSEA-4), and Oct
 4. 60. The composition of claim 49, wherein at least 90% of the embryonic stem cells express one or more of tumor rejecting antigen (TRA), stage specific embryonic antigen-4 (SSEA-4), and Oct
 4. 61. The composition of claim 49, wherein at least 95% of the embryonic stem cells express one or more of tumor rejecting antigen (TRA), stage specific embryonic antigen-4 (SSEA-4), and Oct
 4. 62. The composition of claim 49, wherein at most 10% of the embryonic stem cells express one or more of CD31, alpha-fetoprotein, and tubulin.
 63. The composition of claim 49, wherein at most 5% of the embryonic stem cells express one or more of CD31, alpha-fetoprotein, and tubulin.
 64. The composition of claim 49, wherein at most 1% of the embryonic stem cells express one or more of CD31, alpha-fetoprotein, and tubulin.
 65. The composition of claim 49, wherein the cells encapsulated within the biocompatible matrix maintain a stable phenotype in culture at least 30 doublings.
 66. The composition of claim 49, wherein the cells encapsulated within the biocompatible matrix maintain a stable phenotype in culture for at least 30 days.
 67. The composition of claim 49, wherein the cells encapsulated within the biocompatible matrix maintain a stable phenotype in culture for at least 40 days.
 68. A method of culturing embryonic stem cells, comprising: providing a population of embryonic stem cells; combining the embryonic stem cells with hyaluronic acid to form a mixture; and causing the hyaluronic acid to cross-link in a solvent, thereby encapsulating the embryonic stem cells in a hyaluronic acid hydrogel.
 69. The method of claim 68, further comprising culturing the encapsulated embryonic stem cells in vitro.
 70. The method of claim 68, further comprising maintaining the embryonic stem cells in culture for at least 30 days, and wherein the cells maintain a stable phenotype.
 71. The method of claim 69, further comprising maintaining the embryonic stem cells in culture for at least 40 days, and wherein the cells maintain a stable phenotype.
 72. The method of claim 69, further comprising maintaining the embryonic stem cells in culture for at least 30 doublings, and wherein the cells maintain a stable phenotype.
 73. The method of claim 68, wherein causing comprises promoting radical chain polymerization, ionic chain polymerization, or step polymerization.
 74. The method of claim 68, wherein the hyaluronic acid is terminated with methacrylate groups.
 75. The method of claim 68, wherein the hyaluronic acid is terminated with acrylate groups, thiols, or amines.
 76. The method of claim 68, wherein the molecular weight of the hyaluronic acid is about 50 kDa, about 350 kDa, or about 1100 kDa.
 77. The method of claim 68, wherein the molecular weight of the hyaluronic acid is from about 5 kDa to about 50 kDa.
 78. The method of claim 68, wherein the molecular weight of the hyaluronic acid is from about 50 kDa to about 100 kDa.
 79. The method of claim 68, wherein the molecular weight of the hyaluronic acid is from about 100 kDa to about 500 kDa.
 80. The method of claim 68, wherein the molecular weight of the hyaluronic acid is from about 500 kDa to about 1000 kDa.
 81. The method of claim 68, wherein the molecular weight of the hyaluronic acid is from about 1000 kDa to about 1500 kDa.
 82. The method of claim 68, wherein the molecular weight of the hyaluronic acid is from about 1500 kDa to about 2000 kDa.
 83. The method of claim 68, further comprising allowing the cells to proliferate, releasing the cells from the hydrogel, dividing the cells into a plurality of populations, and repeating the method of claim 68 with each population in the plurality of populations.
 84. The method of claim 68, further comprising contacting the hydrogel with hyaluronidase to release the embryonic stem cells.
 85. A method of producing a population of embryonic stem cells, comprising: providing a population of mammalian embryonic stem cells; combining the embryonic stem cells with methacrylate-terminated hyaluronic acid; causing the hyaluronic acid to cross-link in a solvent, thereby encapsulating the embryonic stem cells in a hyaluronic acid hydrogel; and contacting the hydrogel with hyaluronidase to release the embryonic stem cells.
 86. The method of claim 85, further comprising, before contacting the hydrogel, culturing the encapsulated embryonic stem cells in vitro.
 87. The method of claim 85, further comprising repeating combining and causing with the released embryonic stem cells. 